Study on sesquiterpenoids from Aquilaria sinensis / 中草药

Objective: To investigate the sesquiterpenoid constituents from Aquilaria sinensis. Methods: The chemical constituents were isolated and purified by various separation techniques such as silica gel, ODS, Sephadex LH-20, and preparative high- performance liquid chromatography, and their structures were determined according to their physicochemical properties, MS, 1D, and 2D NMR. The antibacterial activity of the obtained compounds against methicillin-resistant Staphylococcus aureus was tested by 96-well plate microdilution method. Results: Seven sesquiterpenoids were obtained from 95% ethanol aqueous extract of Aquilaria sinensis and their structures were identified as (+)-4a,5-dimethyl-3-(prop-1-en-2yl)-octahydronaphthalene-2β,8a-diol (1), baimuxinic acid (2), baimuxinol (3), vetaspira-2(11),6-dien-14-al (4), baimuxinal (5), (-)-10-epi-γ-eudesmol (6), and 9β-hydroxyl-α-agarofuran (7). The minimum inhibitory concentration (MIC) of compound 1 against methicillin-resistant Staphylococcus aureus was 210 μmol/L. Conclusion: Compound 1 is a new compound named as 2β,8aα-dihydroxy-11-en-eremophilane, which has a good inhibitory effect against methicillin-resistant Staphylococcus aureus.

Differential expression of transcription factors in three generations of excised roots of Aquilaria sinensis under salt stress / 中草药

Objective: To study the expression patterns and levels of transcription factors (TFs) in three generations of excised roots of Aquilaria sinensis under salt stress, and analyze the variation of TFs family genes in each generation in response to salt stress. Methods The excised roots of A. sinensis were used as experimental material, using highthroughput sequencing technology (Illumina Hiseq4000), all the unigene sequences were compared with the plant transcription factor database (PlantTFDB), and the three generation differential expressed TFs between the treated and the control group were analyzed. Results:A total of 48 286 Unigenes were obtained by de novo splicing from three generation of excised roots of A. sinensis, containing 1 156 potential TFs distributed in 54 TF families. Among them, bHLH, ERF, and NAC were the three most enriched families. Totally, 290, 277, and 349 differentially expressed TFs were respectively screened in three successive generations, which were mainly down-regulated. The expression induced by salt stress were different in each TF family, the numbers of up-regulated DEGs increased in NAC, MYB, and WRKY families, and decreased in GRAS family with the increase of stressed generations. There were 70 common TFs differentially expressed in three generations, and the down-regulated expression multiples of eight genes increased with the increase of salt stress generations. Conclusion:The effect of salt stress on the expression of TFs was mainly down-regulatied. The number of differential expressed TFs in the treated and control group increased with the increase of salt stress generations. The expression of different TF family genes was different under salt stress, and some genes might be involved in the transmission of stressful memory. This study is helpful to understand the expression characteristics of TFs at the whole level and provide a reference for further study on the stressful memories and the molecular mechanism of the salt stress response.

Molecular cloning, prokaryotic expression and characterization of a transcription factor AsWRKY62 in Aquilaria sinensis / 中草药

Objective: The molecular cloning and prokaryotic expression of the transcription factor AsWRKY62 of Aquilaria sinensis were carried out, at the same time, the bioinformatics analysis and expression pattern analysis were also performed. The purpose of this study was to lay a foundation for further study on the role of AsWRKY62 in the growth and development of A. sinensis and the formation of agarwood. Methods: With the cDNA isolated from A. sinensis callus as template, the full-length coding sequence (CDS) of AsWRKY62 was amplified using RT-PCR and PCR method. The recombinant vector pET-21a-AsWRKY62 which was built and verified by gene recombination technique was transformed into E. coli BL21 (DE3) for prokaryotic expression and purification. The characteristics of physiochemical properties, conserved domains and subcellular localization of AsWRKY62 were calculated by a series of bioinformatics tools. The analyses of multiple sequence alignment of amino acid and phylogenetic tree were performed using DNAMAN and MEGA 5.0, respectively. The gene expression pattern in different tissues was detected by RNA-seq data. Results: The full length CDS of AsWRKY62 (GenBank accession MH925301) was 1581 bp, encoding a 526-aa protein which belongs to WRKY group I. The optimized induction conditions of recombinant pET-21a-AsWRKY62 were 0.5 mmol/L IPTG at 37 ℃ for 4 h. According to the tissue-specific expression pattern analysis, the AsWRKY62 gene in A. sinensis is mainly expressed in roots and stems, followed by agarwood and flowers. Conclusion: Cloning, expression and characterization of the AsWRKY62 gene for the first time indicated that it may be related to the formation of agarwood, which provided a theoretical basis for further study of its biological function.

Full-length cDNA cloning of AsJAZ1 gene and damage-induced expression analysis in Aquilaria sinensis callus / 中草药

Objective: To clone the full-length cDNA of jasmonate-zim-domain protein (JAZ) gene in Aquilaria sinensis to provide the basic information for further study on gene function in sesquiterpenes biosynthesis pathway. Methods: With the total RNA as template, the full-length cDNA of JAZ in A. sinensis was cloned through rapid amplification of cDNA ends (RACE) technique and reverse transcription PCR (qRT-PCR) method. The bioinformatics of the JAZ gene was analyzed as well. The expression of this gene was detected by qRT-PCR method with MeJA and mechanical wounding treatment in A. sinensis callus. Results: The full-length cDNA (1 507 bp) of JAZ gene was named AsJAZ1; GenBank registration number was KP677281. AsJAZ1 was obtained with an open reading frame (ORF) of 990 bp and encoding 330 amino acids. The relative molecular mass of AsJAZ1 calculated was 34 280, and the isoelecric point was 6.89. Real time PCR results indicated that both MeJA treatment and mechanical wounding could stimulate the increase of mRNA expression of AsJAZ1; There was a sharp rise at 0.5 h with about 27 times higher than the control (without MeJA treatment) with MeJA treatment, then dropped significantly. In mechanical wounding treatment, the highest peak presented in 2 h about 17 times compared to the control, then dropped significantly too. The expression of AsJAZ1 gene returned to be normal in 24 h. Conclusion: We have obtained the full-length cDNA sequence of AsJAZ1 gene firstly, which was extremely sensitive to wounding and responded to the early damage.

Analysis of Fungi Diversity in Agarwood Wood from Hainan Province and Guangdong Province / 中国药学杂志

OBJECTIVE: To characterize the fungal communities in agarwood wood from Hainan province and Guangdong province. METHODS: Fungal ITS genes in the agarwood wood were analyzed by high-throughput pyrosequencing using Illumina HiSeq system and bioinformatically. RESULTS: The fungal microbiomes exhibited much similarity among the samples from different regions in each province. And there were notable differences between Guangdong province and Hainan province. Moreover, the fungal communities in the agarwood wood from Hainan Province showed higher diversity and richness than that in the agarwood wood from Guangdong province. At the phylum level, Ascomycota most dominated in the agarwood wood, followed by Basidiomycota. Ascomycota dominated in the agarwood wood from Haikou, Wanning, Ledong, Danzhou and Dongguan regions. Basidiomycota dominated in the agarwood wood from Huazhou region. At the class level, agaricomycetes dominated in the agarwood wood from Huazhou region. Sordariomycetes dominated in the agarwood wood from Dondguan and Danzhou regions, Dothideomycetes dominated in the agarwood wood from Haikou, Ledong and Wanning regions. At the order level, Pleosporales dominated in the agarwood wood from Haikou, Ledong, Wanning and Dondguan regions. Trechisporales and Polyporales dominated in the agarwood wood from Huazhou and Danzhou regions, respectively. At the genus level, Lignosphaeria dominated in the agarwood wood from Haikou and Wanning regions, Perenniporia and Pyrigemmula dominated in the agarwood wood from Ledong and Danzhou regions, respectively. Phaeoacremonium dominated in the agarwood wood from Huazhou and Dondguan regions. Agarwood wood from different regions contained different dominate fungal populations. CONCLUSION: The fungi in the agarwood wood from Hainan province has higher diversity and richness than that in the agawood wood from Guangdong province. Agarwood wood from different regions has distinct fungal communities.

Molecular Cloning and Expression Analysis of the Transcription Factor AsWRKY25 in Aquilaria sinensis (Lour.) Gilg / 中国药学杂志

OBJECTIVE: To clone AsWRKY25, a transcription factor gene of Aquilaria sinensis, for bioinformatic analysis and tissue expression analysis, and express its protein in prokaryotic cells, thus to lay a foundation for further study of the biological function of AsWRKY25. METHODS: With the cDNA isolated from A. sinensis callus as template, the full-length coding sequence (CDS) of AsWRKY25 was amplified using PCR method. The recombinant vector pET-28a-AsWRKY25 was transformed into Escherichia coli BL21 (DE3) for prokaryotic expression. The physiochemical properties and bioinformatic characters of AsWRKY25 were calculated by a series of bioinformatics tools and softwares. The expression patterns in different tissues were detected by RT-PCR. RESULTS: The full-length coding sequence of AsWRKY25 transcription factor was cloned from the callus of A. sinensis. The CDS of AsWRKY25 was 1 728 bp in length and contained a 1 728 bp open reading frame (ORF) encoding 575 amino acids. The CDS sequence was codon-optimized and then ligated into the pET-28a expression vector. The optimal induction condition of recombinant pET-28a-AsWRKY25 was 1 mmol•L-1 IPTG at 37℃ for 6 h in E. coli BL21 (DE3). The result of tissue expression analysis showed that AsWRKY25 had the highest expression level in the agarwood layer and the lowest level in roots, stems and branches. CONCLUSION: The WRKY transcription factor AsWRKY25 is successfully cloned from A. sinensis and expressed in E. coil. It is speculated that AsWRKY25 may be involved in the wound-induced agarwood-formation process in A. sinensis.

Prokaryotic expression of Aquilaria sinensis AsMAPK3 gene and its localization in transient expression system in onion epidermis / 中草药

Objective To construct a prokaryotic vector of AsMAPK3 gene from Aquilaria sinensis, the original plant of agarwood, and induce the recombinant proteins expression so as to study the subcellular localization of AsMAPK3. This work will prepare materials for antibody preparation and lay a foundation for screening the interaction proteins and further studying their functions. Methods Partial cDNA sequence was amplified by PCR and recombined to pET-28a vector to construct a prokaryotic expression vector pET-28a-AsMAPK3, and induced the expression of the fusion protein. The full-length cDNA of AsMAPK3 was amplified and subcloned to pAN580 vector to construct a pAN580-AsMAPK3 transient expression vector. The recombinant plasmid of pAN580-AsMAPK3 was introduced into the onion epidermis by gold particle bombardment, and GFP fluorescence was observed by luorescence microscope. Results The Escherichia coli BL21 (DE3) containing the recombinant plasmid was induced with 0.5 mmol/L isopropyl-β-D-galactoside (IPTG) at 37 ℃ for 4 h, and a fusion protein about 39 000 was obtained which was expressed in supernatant and inclusion bodies. The results of GFP fluorescence observation of transient transformed onion epidermis showed that AsMAPK3 was mainly expressed in the nucleus and plasma membrane. Conclusion The expression and purification of AsMAPK3 in vitro were successfully carried out, and the subcellular localization of AsMAPK3 gene was confirmed. This work provides a substantial foundation for follow-up function study of AsMAPK3.

Cloning and expression analysis of two small heat shock proteins genes from Aquilaria sinensis / 中草药

Objective: To aim at cloning the open reading frame (ORF) of sHSP1 and sHSP2 genes from Aquilaria sinensis and analyzing the bioinformatics and expression of the two genes. Methods: Two unique sequences containing sHSPs domain were discovered in transcriptome dataset of A. sisnensis. The full-length cDNAs of sHSP1 and sHSP2 were cloned by RT-PCR strategy with the specific primers. Subcellular localization, transmembrane domain, three-dimensional structure, and phylogenetic analysis were predicted by different softwares to analyze the bioinformatics of sHSPs protein. The expression different levels of sHSP1 and sHSP2 isoforms in different tissues and in responds to salt and ABA, SA, MJ treatment were measured by real-time quantitative PCR. Results: The sHSP1 and sHSP2 cDNA sequence consisted of 474 bp ORF, encoding 157 amino acids. Tissue expression analysis indicated that sHSP1 and sHSP2 were primarily expressed in roots, followed by stems and leaves. Salt treatment experiments indicated that salt treatment caused a rapid increase in sHSP1and sHSP2 expression within 36 and 24 h, respectively. Exogenous ABA and MJ treatment experiments indicated that sHSP1 and sHSP2 genes were induced by exogenous ABA and MJ, and all reached the highest expression level at 12 h. Simultaneously, the SA treatment experiments indicated that exogenous SA treatment caused a rapid increase in sHSP1 and sHSP2 expression within 12 and 24 h, respectively. Conclusion: The full-length cDNA sequence of sHSP1 and sHSP2 genes from A. sinensis is obtained. sHSP1 and sHSP2 have the different expression level in different tissues. When subjected to high salt, ABA, SA, and MJ treatment, sHSP1 and sHSP2 show the different expression levels in different time. Cloning and analyzing sHSP1 and sHSP2 genes from A. sinensis will play an important role for further study on plant defense response.

Relationship between expression of sesquiterpene synthase gene and sesquiterpene content in artificial agarwood induced by Fusarium sp. A2 / 中国药学杂志

OBJECTIVE: To explore the expression of sesquiterpene synthase (gene sesqui-TPS) and dynamic change of sesquiterpene content in the resin-deposited parts of the trunk of Aquilaria sinensis (Lour.) Gilg. induced by Fusarium sp. A2. METHODS: Artificial agarwoods from Aquilaria sinensis were induced by Fusarium sp. A2 using pinhole instillation. The heartwood of Aquilaria sinensis without or with resin were extracted before induction and at 7 time points within one year after induction. The expression of sesqui-TPS was detected by quantitative Real-time PCR. And then the dynamic change of sesquiterpene content in the tissues were analyzed by gas chromatography-mass spectrometry (GC-MS). RESULTS: The relative expression of sesqui-TPS gene in the artificial agarwoods induced by Fusarium sp. A2 from 2 to 12 months were 10.85, 0.793 1, 6.484, 611.4, 5 800, and 4 211 respectively. Sesquiterpene secondary metabolites were not detected in the trunk at 2 months before artificial induction. Fourteen sesquiterpene were detected from 4 to 12 months, with relative percentages of 21.40%, 25.52%, 36.44%, 32.40% and 55.70% at each time point. CONCLUSION: Sesqui-TPS gene is extremely sensitive to Fusarium sp. A2 treatment and responds to late damage. The expression of sesqui-TPS gene lags behind the accumulation of sesquiterpene component, which would provide a foundation for studies on regulation action of function gene for sesquiterpene biosynthase pathway during the formation of agarwood resin in A. sinensis.

Structural elucidation of two new megastigmane glycosides from the leaves of Aquilaria sinensis / 中国天然药物

The present study was designed to determine the chemical constituents and identify new components of the leaves of Aquilaria sinensis (Lour.) Gilg. The compounds were isolated and purified by repeated silica gel, Sephadex LH-20, and ODS column chromatography and their structures were elucidated by NMR and HR-ESI-MS spectrometry. Eight megastigmane glycosides and two cucurbitacins were isolated and identified as (9S) megastigma-4,7-diene-2,3,9-triol 9-O-β-D-glucopyranoside (1), (9S) megastigma-4(13),7-diene-3,6,9-triol 9-O-β-D-glucopyranoside (2), macarangloside D (3), corchoionoside C (4), staphylionoside H (5), (+) 3-oxo-α-ionol-β-D-glucopyranoside (6), (-) 3-oxo-α-ionol-β-D-glucopyranoside (7), citroside B (8), 2-O-β-D-glucopyranosyl cucurbitacin I (9), bryoamaride (10). Compounds 1 and 2 were newly identified megstigmane glucosides and reported from this genus for the first time.

Cloning, bioinformatics, and expression analysis of sesquiterpene synthase gene As-SesTPS1 from Aquilaria sinensis / 中草药

To clone a sesquiterpene synthase gene denoted As-SesTPS1 from total RNA of Aquilaria sinensis based on previous transcriptome sequencing data and analyze the bioinformatics and expression of the gene. The full length cDNA of As-SesTPS1 was obtained by reverse transcription-PCR (RT-PCR). The sequence similarity and homology were analyzed, and the structure and function of the coding protein were predicted by bioinformatics method. The gene expression levels in different samples of A. sinensis were quantified using qRT-PCR technique. The sesquiterpene synthase gene As-SesTPS1 was cloned, which contained an ORF of 1 671 bp, encoding 556 amino acids, and shared high similarity to multiple sesquiterpene synthase genes. The protein encoded by this gene had conserved RRx8W and DDxxD motifs. It was confirmed the gene was highly expressed in agarwood sample through qRT-PCR technique. The sesquiterpene synthase gene As-SesTPS1 is cloned, which will lay a foundation for further study of sesquiterpene biosynthase pathway in A. sinensis.

Full-length cDNA cloning of AsNINJA1 gene in Aquilaria sinensis and MeJA-induced expression in callus / 中草药

Objective: To clone the full-length cDNA of interacting protein of JAZ (NINJA) gene in Aquilaria sinensis, and to provide the basic information for further study on gene function in sesquiterpenes biosynthesis pathway. Methods: With the total RNA as template, the full-length cDNA of NINJA in A. sinensis was cloned through RACE technique and RT-PCR method. The bioinformatics of cloing NINJA gene was analyzed as well. The expression mode of this gene was with MeJA treatment in A. sinensis callus detected by qRT-PCR method. Results: The full-length cDNA (1 982 bp) of NINJA gene in A. sinensis, named as AsNINJA1 was obtained with an open reading frame of 1 221 bp and encoding 406 amino acids. The relative molecular mass of AsNINJA1 protein calculated was 43 697, and the isoelectric point was 6.02. The qRT-PCR results indicated that MeJA treatment could stimulate the increase of mRNA expression of AsNINJA1; There was a sharp rise at 4 h with nearly 100 times higher than the control (without MeJA treatment), then dropped significantly. Conclusion: The full-length cDNA sequence of AsNINJA1 gene is obtained; AsNINJA1 is extremely sensitive to MeJA treatment, and responded to the early damage.

Chemical constituents from pericarps of Aquilaria sinensis and their biological activities / 中草药

Objective: To study the chemical constituents from the pericarps of Aquilaria sinensis and their cytotoxic and antibacterial activities. Methods: The compounds were isolated by various chromatographic methods (silica gel, reverse silica gel, Sephadex LH-20, preparative TLC, and so on) and recrystallization. Their structures were identified by extensive analysis of the spectroscopic data. The cytotoxic effects of these compounds on neuroglioma SF-268, breast cancer MCF-7, large cell lung cancer NCI-H460, and hepatoma carcinoma HepG-2 cell lines were observed in vitro by SRB staining, and the antibacterial effects of these compounds on Staphyloccocus aureus and Escherichia coli were observed in vitro by Resazurin staining method. Results: Ten compounds were isolated from the pericarps of A. sinensis and identified as isorhamnetin 3-O-(6″-O-(Z)-p-coumaroyl)-β-D-glucopyranoside (1), buddlenoid A (2), 7-methoxy-4'-hydroxyisoflavone (3), luteolin (4), trans-p-coumaric acid ethyl eater (5), costunolide (6), epifriedelanol (7), stigmasterol (8), p-hydroxybenzoate (9), and pyrocatechol (10). Conclusion: Compounds 1-3, 5, 6, 9, and 10 are first isolated from the plant and compounds 1-3, 5, and 6 are isolated from the plants of genus Aquilaria Lam. for the first time. Compound 6 exhibits the significant cytotoxic activities against the above four cell lines and compounds 1 and 4 show some antibacterial activities against S. aureus and E. coli.

Fungus inducing the formation of agilawood from Aquilaria sinensis / 中国药学杂志

OBJECTIVE: To screen the fungi which can induce Aquilaria sinensis to produce active substance. METHODS: Fungus was inoculated in the stem of A. sinensis under natural conditions. The penetration and planting of fungus in stem of A. sinensis as well as the interaction of the two organisms were studied by microscope and scanning electron microscopy, and the main ingredients of the volatile oil in induced agilawood were analysed by GC-MS. RESULTS: Three active strains inducing the formation of agilawood were obtained, which was reported for the first time in the world. The main ingredients of the volatile oil were similar between the induced agilawood and the natural materials. CONCLUSION: It is very important for us to use the fungus in the induction of agilawood and in the sustainable utilization of this special medicinal material. Copyright 2012 by the Chinese Pharmaceutical Association.

Study on Cryopreservation of Seeds of Aquilaria sinensis(Lour.)Gilg / 广州中医药大学学报

【Objective】To explore the long-term storage method for the seeds of Aquilaria sinensis(Lour.)Gilg..【Methods】The seeds of Aquilaria sinensis(Lour.)Gilg.with different water contents were preserved with liquid nitrogen for 55 days.And then the seeds were taken out to detect their germination capacity.The effect of cryopreservation methods on the germination capacity was also observed.【Results】The seeds with water content being 16.30% lost their germination capacity completely,the seeds with water content being 13.02% and 9.34% had a lower germination capacity,and the seeds with water content being 7.35% had a higher germination capacity.Quickly-frozen seeds had a higher germination capacity than the slowly-frozen seeds.【Conclusion】Cryopreservation with liquid nitrogen is suitable for the long-term preservation of the seeds of Aquilaria sinensis (Lour.)Gilg.with water content being 7.35%.

Induction of chromones in excised lateral roots culture of Aquilaria sinensis / 中草药

Objective To study the biosynthesis of the second metabolites in Aquilaria sinensis.Methods Chromones of agarwood were elicited by tissue culture and the inducer contents were determined by HPLC.Results 2-(2-Phenylethyl) chromones could be elicited in excised lateral roots suspension culture of A.sinensis.Conclusion Fungal extracts of Menanotus flavolives could induce the production of characteristic components of agarwood in A.sinensis.